The value of multi-modal gene screening in HNPCC in Quebec: three mutations in mismatch repair genes that would have not been correctly identified by genomic DNA sequencing alone.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by a mutation in one of the mismatch repair genes, most frequently MLH1 or MSH2. The rate of mutation detection is influenced by many factors, including the diagnostic methods used. Large deletions, which occur frequently in MLH1 and MSH2, are not detected by exon-by-exon screening methods. Here, we describe three mutations in mismatch repair genes detected using a screening protocol that combines protein truncation test (PTT) analysis and multiplex ligation-dependent probe amplification (MLPA) with genomic and cDNA sequencing. Two of these mutations consist of large deletions in MLH1 that were detected by both MLPA and PTT but that would have been missed by genomic DNA sequencing. The third is a large deletion in MSH2 that could not be detected by PTT because of its location relative to the primers used to amplify the cDNA, or by sequencing. This mutation was detected by MLPA.

Distinct patterns of germ-line deletions in MLH1 and MSH2: the implication of Alu repetitive element in the genetic etiology of Lynch syndrome (HNPCC).

A relatively high frequency of germ-line genomic rearrangements in MLH1 and MSH2 has been reported among Lynch Syndrome (HNPCC) patients from different ethnic populations. To investigate the underlying molecular mechanisms, we characterized the DNA breakpoints of 11 germ-line deletions, six for MLH1 and five for MSH2. Distinct deletion patterns were found for the two genes. The five cases of MSH2 deletions result exclusively from intragenic unequal recombination mediated by repetitive Alu sequences. In contrast, five out of the six MLH1 deletions are due to recombinations involving sequences of no significant homology (P=0.015). A detailed analysis of the DNA breakpoints in the two genes, previously characterized by other groups, validated the observation that Alu-mediated unequal recombination is the main type of deletion in MSH2 (n=34), but not in MLH1 (n=21) (P<0.0001). Plotting the distribution of known DNA breakpoints among the introns of the two genes showed that, the highest breakpoint density is co-localized with the highest Alu density. Our study suggests that Alu is a promoting factor for the genomic recombinations in both MLH1 and MSH2, and the local Alu density may be involved in shaping the deletion pattern.